DE3780043T3 - Penicillium chrysogenum. - Google Patents

Description

This invention relates to Penicillium chrysogenum.

The ascomycete P. chrysogenum is a filamentous fungus used for the industrial production of penicillins. Much research has been done to obtain high-yielding strains using classical mutagenesis and forced selection.

An object of this invention is to apply the molecular cloning method to P. chrysogenum. In particular, this invention is directed to developing transformation into P. chrysogenum with exogenous DNA, with a view to providing the possibility of applying genetic engineering to obtain high-yield penicillin-producing strains of P. chrysogenum.

The subject invention enables the use of auxotrophic mutants of P. chrysogenum as receptors for exogenous DNA. The auxotrophic mutant lacks a biosynthetic ability of the wild type of P. chrysogenum. Successful restoration of this biosynthetic ability for prototrophy can be used as a marker for the incorporation of an exogenous DNA. For this purpose, the exogenous DNA is a DNA that includes a DNA that complements the existing DNA of the auxotrophic mutant to restore the missing biosynthetic ability.

The auxotrophic markers of interest include trpC, pyr4, argB and NO⊃min;3 reductase.

PREFERRED DESIGNS

The subject invention enables, for example, the use of wild-type P. chrysogenum biosynthetic genes for the selection and identification of clones with incorporated foreign DNA. This exogenous DNA can include wild-type P. chrysogenum DNA to restore prototrophic reproduction.

For example, the marker can be based on the tryptophan biosynthetic pathway genes of wild-type P. chrysojenum, such as the wild-type trpC gene. Through the present work, we have demonstrated that the biosynthesis of tryptophan in P. chrysogenum is similar to that in other filamentous fungi, such as Neurospora crassa or Aspergillus nidulans, and depends on a multifunctional protein complex, anthranilate synthetase, which also has phosphoribosyl anthranilate isomerase and indole glycerol phosphate synthetase activities. The trpC gene encodes a subunit of this protein, which has the latter two activities in addition to the glutamine amido-transferase activity of anthranilate synthetase.

Thus, according to an example of the subject invention, due to an ineffective trpC gene, the mutant is trp⊃min; and the exogenous DNA complements the existing DNA to restore the trpC gene and yield a prototroph.

In general, suitable wild-type P. chrysogenum DNA includes plasmids that can transform the auxotrophic mutant to restore the desired activity. Continuing the example of the trp⊃min; mutants, the plasmid can be a plasmid that restores a wild-type trpC gene. Specific plasmids with this ability are plasmids pPctrpC1 and pPctrpC6, which contain a suitable insert at the unique BamHI site of the known plasmid pUC13. Escherichia coli containing pPctrpC1 and Escherichia coli containing pPctrpC6 were deposited in accordance with the Budapest Treaty on 14 March 1986 in the National Collection of Industrial Bacteria, Scotland, and registered under the number NCIB 12222 and NCIB 12223 respectively.

The plasmids, such as the plasmids pPctrpC1 or pPctrpC6, can themselves be identified by their ability to complement a deficient mutant of Escherichia coli, for example the known trp⊃min; E. coli mutant trpC9830.

A particularly preferred tryptophan-requiring mutant of P. chrysogenum as a host strain is the mutant P. chrysogenum ATCC10003 trp2. This strain was deposited at the Commonwealth Mycological Institute, England, under the Budapest Treaty on 20 March 1986 and registered under the number CMICC302709.

It should be noted that the subject invention is not limited to the restoration of tryptophan biosynthesis in trp⊃min; P. chrysogenum by transformation. Other illustrative examples of auxotrophic mutants include those with incomplete pyr4, argB, or NO⊃min;3 reductase genes.

The invention is explained in more detail with reference to the accompanying drawings using the following examples, in which:

Fig. 1 the procedure according to Example 1 for the cloning of the phosphoribosyl anthranilate isomerase complementing activity of P. chrysogenum;

Fig. 2 the restriction maps of six plasmids obtained according to Example 3, as well as the associated information; and

Fig. 3 a more complete restriction map for one of the six plasmids in Fig. 2.

EXAMPLES OF THE INVENTION Dining room 1 Molecular cloning of the trpC gene

As illustrated in Fig. 1, DNA from P. chrysogenum ATCC10003 was partially digested with restriction endonuclease Sau3AI, resulting in a mixture of fragments with an average size of 5 to 10 Kb. The digestion mixture was layered on a 5 to 24% NaCl gradient in 3 mM EDTA, pH 8.0 and centrifuged for 19 hours at 17,000 rpm in a SW40 rotor. The gradient was fractionated and samples from each fraction were analyzed by separating in 0.8% agarose gel with standards of appropriate molecular weight. Fractions containing DNA fragments of 8 to 10 Kb were pooled, dialyzed against TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) and concentrated by ethanol precipitation.

Separately, the plasmid pUC13 (Methods in Enzymology (1983) 101, 20-78) was completely digested with BamHI. After inactivation of the enzyme by phenol extraction, the phosphate groups of the 5' ends were eliminated by treatment with 0.04 units of alkaline phosphatase for 30 minutes at 37°C to prevent self-ligation of the vector. The second enzyme was inactivated by repeated phenol extraction and the DNA was concentrated by phenol precipitation.

The phosphatase-treated DNA from the pUC13 and the P. chrysogenum fragments of 8 to 10 Kb were mixed at a molar ratio (vector:insert) of 10:3 and incubated overnight with 6 units of DNA ligase at 14ºC. The resulting ligated DNA was used to generate E. coli strain HB101 (J. Mol. Biol. (1969) 41, 459-472). to ampicillin resistance. A total of 310,000 transformants were obtained. Analysis of a statistically significant number of transformants showed that approximately 60% of the colonies contained recombinant plasmids and that the average insert size was approximately 8 Kb. Plasmid DNA from these 310,000 transformants was purified by alkaline lysis followed by CsCl-EtBr equilibration centrifugation. The purified DNA was stored at -80ºC until use.

The trpC9830 mutation of E. coli strain MC1066 (J. Molec. Appl. Genet. (1983) 2, 83-99) [lac(IPOZYA)X74, galK, galU, strA, hsdR, trpC9830, leuB6, pyrF74:Tns(Kmr)] was used as a host for the selection of E. coli trpC by complementation. The amount of DNA used was large enough to obtain 358,000 transformants with an average insert size of 8 Kb, representing 16 times the genome of P. chrysogenum. Cells were plated on selective medium lacking tryptophan. After 1 to 9 days of incubation at 37ºC, eleven colonies with the desired pHenotype (trp+, ampr) were observed. These colonies of the plasmids containing the insert were inoculated into small cultures and the plasmid DNA was purified.

Plasmid DNA from these colonies was transformed into E. coli HB101 and selected transformants were grown in large scale cultures. The restriction maps for 6 of these plasmids, designated plasmid pPctrpC1 to pPctrpC6, are shown in Fig. 2. All of these plasmids complement the phosphoribosyl anthranilate isomerase deficiency when reintroduced into E. coli MC1066.

The largest insert of 16 Kb is contained in plasmid pPctrpC6. All other inserts in the other plasmids are contained in plasmid pPctrpC6, and the restriction maps in the overlapping regions are identical, indicating that no cloning artifacts Furthermore, genomic clones were isolated from a P. chrysogenum library constructed in lambda EHBL4 using pPctrpC4 as a probe. The restriction maps of these phage clones were completely identical to those of the plasmids in the overlapping regions, demonstrating that the true configuration of the P. chrysogenum DNA region is shown in the drawing.

The six plasmids were labeled using nick-resynthesis 32p and hybridized into restriction enzyme digests of P. chrysogenum DNA. The hybridization patterns clearly indicated the presence of P. chrysogenum DNA inserts.

A restriction map for the plasmid p{ctrpC1 is shown in Fig. 3.

Example 2 Construction of the P. chrysogenum trp2 mutant

Tryptophan auxotrophs were obtained from spore suspensions of P. chrysogenum ATCC10003 that had been mutagenized with UV light to a survival rate of approximately 1% of the original cell number. Colonies were streaked onto complete medium plates supplemented with 250 g/ml tryptophan and replicated onto minimal medium plates with or without tryptophan. Colonies exhibiting the trp phenotype were selected for further experiments. They all grew well in minimal medium plus tryptophan, but only formed spores when indole (50 µg/ml) was added. One of these mutants, designated trp2, was particularly promising for the following reasons:

(1) It proliferated very vigorously when tryptophan (250 µg/ml) was added and sporulated well when indole (50 µg/ml) was added.

(2) It did not grow in minimal medium plus anthranilic acid, but grew in minimal medium supplemented with indole. This is the expected phenotype for mutants lacking phosphoribosyl-anthranilate isomerase activity.

(3) It can be transformed into the trp+ phenotype by plasmids containing at least the genetic information encoding the wild-type phosphoribosyl-anthranilate isomerase activity of P. chrysogenum.

(4) It exhibits a low reversion frequency to tryptophan prototrophy (less than 10⁻⁷).

Example 3 Transformation of P. chrysogenum ATCC10003 trp2 with pPctrpCl or pPctrpC6.

The trp2 mutant from Example 2 was propagated in semi-defined medium (0.6% NaNO3, 0.52% MgSO4.7H2O, 0.52% KCl, 1% glucose, 0.5% yeast extract, 0.5% casaminic acid, FeSO4.7H2O trace, ZnSO4 trace). A one-liter Erlenmeyer flask containing 100 ml of medium was inoculated with 107 spores and incubated at 28°C for 44 hours. The mycelium was recovered by filtration and washed several times with distilled water. One gram of the wet mycelium was resuspended in 10 ml 1.2 M KCl and protoplasts were generated by incubating this suspension with Novozym 234 (20 mg/g mycelium) for 4 to 5 hours with shaking. The protoplasts were separated from the mycelial debris by filtration through glass wool and pelleted by centrifugation at low speed.

For transformation, protoplasts were pelleted by centrifugation and resuspended in 10 mM CaCl₂, 10 mM Tris-HCl pH 8.0, 1.2 M KCl at 10⁹ protoplasts/ml and incubated for 10 minutes at 30ºC. 0.1 ml Samples were mixed with 10 pg of the plasmid DNA (plasmid pPctrpCl or pPctrpC6) and 2 ml of 30% PEG 6000 (w/v), 10 mM CaCl2, 10 ml Tris-HCl pH 8.0, 1.2 M KCl and the mixture was incubated for 5 minutes at room temperature. After recovery by low speed centrifugation, the protoplasts were resuspended in 1 ml of 1.2 M KCl, stabilized with 5 ml of osmotically stabilized minimal medium agar, maintained at 50°C and plated on the same medium. Plates were incubated at 28°C and examined for transformants after 3 to 4 days. Typically, transformants occurred at a frequency of approximately 50 transformants/µg DNA, but only on plates containing protoplast samples exposed to the transforming DNA of plasmids pPctrpC1 or pPctrpC6.

Tryptophan-plus colonies were then taken from the selection plates and propagated in selective minimal medium. Two types of colonies appeared and retained their properties after several passages in minimal medium. One colony type was large and similar in morphology and growth rate to the wild type. The other colony type had smaller colonies and grew more slowly. Southern hybridization with 32p-labeled plasmid pUC13 demonstrated that the trp+ colonies contained transforming DNA. In all clones tested, transformation occurred by integration into the recipient genome, resulting in high stability of the trp+ phenotype.

Example 4 Localization of trpC complementing activity

The minimally overlapping region of the inserts in plasmids pPctrpC1 to ppctrpc6 is shown in the lower part of Fig. 2. By deleting up to the second HindIII site in the right end of the minimally overlapping sequence (plasmid pST115), the complementing activity was not inactivated and this activity is thus localized to a 1.15 Kb fragment. However, the SalI-HindIII fragment missing in the plasmid pSTO85 is essential for complementation. A further nucleotide analysis of the plasmid pPctrpC1 localized the beginning of the trpC gene between the SAII and HindIII sites to the right of the insert.

In a further study, we demonstrated that the protein encoded by the DNA insert in the plasmid pPctrpC1 has the following amino acid sequence:

Claims (11)

1. A selectable transformation method for Penicillium chrysogenum, which comprises first selecting an auxotrophic strain of P. chrysogenum and transforming it with a plasmid containing the DNA of a fully prototrophic strain of P. chrysogenum, said plasmid having been selected by prototrophic transformation of a suitable auxotrophic strain of a host microorganism. 2. The method of claim 1, wherein the marker is based on the tryptophan biosynthetic pathway genes of wild type P chrysogenum. 3. The method of claim 2, wherein the marker is trpC. 4. The method of claim 3, wherein the P. chrysogenum is P. chrysogenum ATCC 10003 trp2, CMICC 302709. 5. A plasmid containing a wild-type gene of a fully prototrophic strain of P. chrysogenum, which plasmid has been selected by prototrophic transformation of a suitable auxotrophic strain of a host microorganism, which plasmid confers the prototrophy of an auxotrophic strain of P. chrysogenum can restore. 6. A plasmid as defined in claim 51 which restores the prototrophy of a trp⊃min; mutant of P. chrysogenum. 7. Plasmid as defined in claim 6, which contains a DNA encoding the following amino acid sequence 8. Plasmid pPctrpC1, NCIB 12222. 9. Plasmid pPCtrpC6, NCIB 12223. 10. P. chrysogenum ATCC 10003 trp2, CMICC 302709. 11. A prototrophic strain of P. chrysogenum obtained by introducing a plasmid containing DNA that complements auxotrophy into an auxotrophic strain of P. chrysogenum, which introduction results in more exogenous plasmid DNA being introduced into the genome than the DNA that restores prototrophy to the strain.